Transepithelial transport in cell culture: Stoichiometry of Na/phlorizin binding and Na/d-glucose cotransport. A two-step,two-sodium model of binding and translocation |
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Authors: | Dayton S Misfeldt Martin J Sanders |
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Institution: | (1) Department of Medicine (Oncology), Palo Alto Veterans Administration Medical Center and Stanford University, 94305 Stanford, California;(2) Present address: Center for Ulcer Research and Education, VA Wadsworth Medical Center, 90073 Los Angeles, California |
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Abstract: | Summary The renal cell line LLC-PK1 cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependentd-glucose coupled transport. The short-circuit current (I
sc) was acccounted for by net apical-to-basolaterald-glucose coupled Na flux, which was 0.53±0.09(8) eq cm–2hr–1, andI
sc, 0.50±0.50(8) eq cm–2hr–1. A linear plot of concurrent net Na vs. netd-glucose apical-to-basolateral fluxes gave a regression coefficient of 2.08. As support for a 2 1 transepithelial stoichiometry, sodium was added in the presence ofd-glucose and the response ofI
sc analyzed by a Hill plot. A slope of 2.08±0.06(5) was obtained confirming a requirement of 2 Na for 1d-glucose coupled transport. A Hill plot ofI
sc increase to addedd-glucose in the presence of Na gave a slope of 1.02±0.02(5). A direct determination of the initial rates of Na andd-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2 A coupling ratio of 2 for Na,d-glucose uptake, doubles the potential energy available for Na-gradient coupledd-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependentphlorizin binding was 1.1±0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of Na]. Although occurring at the same site the process of Na-dependent binding of phlorizin differs from the binding and translocation ofd-glucose. Our results support a two-step, two-sodium model for Na-dependentd-glucose cotransport; the initial binding to the cotransporter requires a single Na andd-glucose, a second Na then binds to the ternary complex resulting in translocation. |
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Keywords: | kidney cell line LLC-PK1 stoichiometry phlorizin binding coupled uptake Hill plot electrophysiology I
sc isotope flux sodium gradient |
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